Subject matter of the invention is a method for the determination of an analyte in a sample, a reagent kit for performing this method and the use of two differently labeled probes for the quantitative determination of an analyte in a sample.
The determination of an analyte in a sample has acquired especial importance particularly in the field of health care. Many analytes e.g in body fluids can be used to provide an indication for the presence of an illness or an infection. Many analytes are however not directly determinable or are present in small quantities alongside a large amount of a very similar substances contained in the sample such that direct detection is practically impossible. For this reason increasingly the specific determination of an analyte is attempted with the aid of detectable, labeled probes. Ideally these probes bind only to the analytes and thus labeling the analyte. In the meantime a plurality of labeled groups are available. These include for example metals, chromophores, fluorescent markers but also enzymes, electroluminescent groups and chemically activatable groups.
In WO 92/14139 for example a device is described for performing a binding test for an analyte to be determined which is based on electrochemiluminescence. In this case an analyte is determined by virtue of its binding to a labeled probe using metal complexes and excitation of the complex by applying an electrical potential. The light signal generated is then detected.
Similarly calcium-activatable photoproteins have been suggested as labels e.g. in EP 764 468. The mechanism of light generation in these proteins is based on the addition of a calcium-salt-containing solution to a solution containing an analyte labeled with a probe which has been labeled with aequorin. This triggers the generation of an electromagnetic signal by aequorin.
Nucleic acids are particularly useful aids in the field of diagnostics because of the information stored in their base sequences. Special nucleic acid sequences however are present in a much lower quantity than very similar sequences. It has therefore proven to be of advantage to amplify the nucleic acids to be detected prior to their detection, i.e. production of a plurality of copies of a certain sequence. The Polymerase Chain Reaction (PCR) (U.S. Pat. No.4,683,202) is such a method. Particularly in this amplification reaction it has been discovered that a quantitative detection of nucleic acids is not possible or is only possible under very favorable conditions because the amplification efficiency is heavily influenced by different factors. Therefore it has been suggested that a standard analyte be added in a known amount to the analyte-containing sample prior to amplification. The standard should differ from the analyte in its detection properties but behave in a similar manner with respect to its amplification efficiency. Such a polymerase chain reaction which makes use of internal standards is for example described in U.S. Pat. No. 5,213,961 or U.S. Pat. No. 5,219,727. Similar methods are described in WO 92/01812, WO 94/04706, EP-A-0 525 882, WO 95/02067 and WO 94/09156. In the last-named patent application a method is described in which an immobilized primer is used in the PCR and the amount of amplification achieved is determined using probes.
In WO 93/10257 a method is described in which firstly PCR is performed and then the reaction mixture incubated with two differently labeled probes.
In WO 89/10552 an apparatus for the simultaneous determination of two samples using different labels which can be excited to electrochemiluminesce is described. Two measuring cells and two detectors are used which generate different wavelengths which are then detected. Method in which the excitation step functions in a manner which is spacially and chronologically separate to guarantee acceptable dynamics and sensitivity have the disadvantage that the sample throughput is small and the amount of sample volume required is large.
DE-C-3022426 describes an immunoassay in which a chemiluminescent label is excited to the point of emission by oxidation products produced by the application of an electrical potential.
In EP-0 478 626 a detection group is described which is modified by various substances in such a manner that the resulting detection group displays different kinetic behavior or exhibits a different spectrum. Excitation occurs simultaneously using the same trigger (oxidative). The signals (spectral or kinetic) in practice overlap to a very large extent such that reduced dynamics and lowered sensitivity result.
In EP-0-199 804, U.S. Pat. No. 5,238,808 and U.S. 5,310,687 a multi-labeling system is described in which the various labels are detected optically making use of their differing spectral characteristic.
In WO 93/01308 a method for detecting an analyte using acridinium ester-labeled antibodies and generation of a chemiluminescent signal by oxidation at an alkaline pH is described.
One object of the present invention was therefore in part or in whole to improve on the state of the art and in particular to make available a method in which the disadvantages of previous simultaneous fluorescence excitation methods (non-resolvable or poorly resolved signals) are avoided and at the same time increasing the throughput of the analyzer.
Subject matter of the invention is therefore a method for the determination of an analyte in a sample by incubation of the sample with at least two probes of which at least one is specific for the analyte to be determined and whereby the at least two probes carry different labeling groups, each being capable of generating a different electromagnetic signal for each different labeling group and evaluation of the signal generated is taken as an in indication of the presence of an amount of the analyte. Another subject matter of the invention is a reagent kit for the determination of an analyte and the application of two probes which can supply two different electromagnetic signals for the quantitative detection of analyte in a sample.